Systems and devices for isothermal biochemical reactions and/or analysis

ABSTRACT

An isothermal reaction and analysis system may include a receiver to receive sample holders, a thermal control subsystem to control a temperature of the receiver, an excitation subsystem, a detection subsystem and an analysis subsystem. Excitation sources and/or detectors are positioned to enhance data collection. Sample holders may include filters, selectively blocking and passing wavelengths or bands of electromagnetic radiation.

BACKGROUND

1. Field

This disclosure generally relates to systems and devices for isothermalbiochemical reactions, for example nucleic acid amplification or cellgrowth, and/or analytic equipment to analyze the products or results ofsuch biochemical reactions.

2. Description of the Related Art

There are a number of known techniques to perform biochemical reactions,for example to amplify nucleic acids such as DNA and RNA.

One of the primary existing techniques is polymerase chain reaction(PCR) method, such as that described in U.S. Pat. No. 4,683,195; U.S.Pat. No. 4,683,202; and U.S. Pat. No. 4,800,159. Another existingtechnique is reverse transcription (RT) PCR. Other techniques includeligase chain reaction (LCR) and transcription-based amplification system(TAS).

These techniques each require repeating a set of reactions for eachsample at two or more distinctly different temperatures, commonly knownas thermal cycling. These techniques typically require strict controlover a wide range of temperatures. There are a number of thermal cyclingdevices on the market. It is commercially desirable that these thermalcycling devices be capable of rapidly adjusting between the desiredreaction temperatures in order to increase the number of reactions perunit of time, thereby increasing throughput and reducing costsassociated with operation of such thermal cycling devices. The need tostrictly control temperature over a wide range with a fast response timecauses these thermal cycling devices to be expensive, for examplecosting between $40,000 and $60,000. Due to these requirements,thermally cycling device are also large, and are not suitable to beingprovided in portable or handheld forms.

Amplification techniques that avoid thermal cycling are becoming morepopular. For example, strand displacement amplification (SDA) such asdescribed in Japanese Examined Patent Application No. JP-B7-114718 andvarious modifications of SDA such as described in U.S. Pat. No.5,824,517 and International Patent Application Nos. WO99/09211;WO95/25180 and WO99/49081. Also for example, self-sustained replication(3SR), nucleic acid sequence based amplification (NASBA) such asdescribed in Japanese Patent No. 2650159, transcription mediatedamplification (TMA), and Q beta replicase method such as described inJapanese Patent No. 2710159. Further examples include loop mediatedisothermal amplification (LAMP) such as described in WO00/28082 andexponential amplification reaction (EXPAR) such as described in U.S.Patent Application Publication Nos. 2003/0082590, 20003/0104431;2003/0138800 and 2003/0165911.

It is common to employ thermal cycling devices to perform isothermalbiochemical reactions, even though thermal cycling is not required forsuch isothermal processes. However, as noted above, devices capable ofthermal cycling are prohibitively expensive. Such devices are alsotypically large, and not portable or suitable to handheld formats.

A number of analytic devices exist to analyze samples that have beensubjected to biochemical reactions such as nucleic acid amplification.Such analytic devices typically employ laser or monochromator basedexcitation systems. Such devices are typically large and expensive.

Commercial acceptance of alternative amplification techniques isdependent on a variety of factors, such as the cost of suitable devices,speed of operation and effectiveness in performing amplification and/oranalysis. Commercial acceptance may also depend on the portability ofisothermal reaction and/or analysis devices. Commercial acceptance mayadditionally, or alternatively be dependent on the ability for anisothermal reaction and/or analysis device to work with existing sampleholders, avoiding the need to stock multiple types of sample holders orthe need to replace existing stocks of sample holders. Therefore, it maybe desirable to have novel biochemical reaction and/or analysis devices.The present disclosure is directed to overcoming one or more of theshortcomings set forth above, and providing further related advantages.

BRIEF SUMMARY

In one aspect, the present disclosure is directed to a system to supportbiochemical reactions that includes a receiver having a plurality ofreceptacles formed therein, each of the receptacles sized to at leastpartially receive and support a respective one of a plurality of sampleholders; an excitation subsystem including at least a first plurality ofexcitation sources positioned at least partially in the receiver todirect electromagnetic energy toward at least a portion of a respectiveone of the sample holders received in a respective one of thereceptacles of the receiver, the excitation subsystem operable to excitewith the electromagnetic energy each of a number of samples contained inrespective ones of a plurality of the sample holders while the sampleholders are received and supported by the receiver; a detectionsubsystem that includes a plurality of detectors, each of the detectorspositioned to detect emission of electromagnetic energy from arespective sample contained in a respective one of the sample holdersthat are received and supported by the receiver; and a thermal controlsubsystem operable to at least approximately maintain a temperature ofthe receiver at least approximately constant for a period of timesufficient to perform an isothermal reaction on the samples. This mayinclude heater and a heat removal mechanism or a cooler or coolingmechanism that may involve passive and/or active cooling approaches. Theexcitation subsystem may include a second plurality of excitationsources, each of the excitation sources of the second plurality ofexcitation sources positioned at least partially in the receiver todirect the electromagnetic energy toward at least a portion of arespective one of the sample holders received in a respective one of thereceptacles of the receiver, the electromagnetic energy directed by theexcitation sources of the second plurality of excitation sources beingof a different wavelength than the electromagnetic energy directed bythe excitation sources of the first plurality of excitation sources. Theexcitation sources may advantageously be positioned in a respectivepassage in the receiver to direct the electromagnetic energy toward onlya base portion of the respective sample holder when the sample holder ispositioned in the respective one of the receptacles, the base portion atleast proximate a bottom of the sample holder when supported by thereceiver. A filter may be positioned between the excitation sources andthe respective sample holders. A filter may be positioned between thedetectors and the respective sample holders, for example to filter atleast a portion of the electromagnetic energy emitted by at least one ofthe excitation sources. The thermal control subsystem may includes atemperature sensor to detect a temperature of a biochemical reaction orthe receiver and may include a heater element operable to provide heatto the receiver and/or a cooling element to withdraw heat from thereceiver in response to a difference between the detected temperatureand a set temperature. In contrast to thermal cycling devices, thereceiver may have a large thermal mass with respect to the samples.

The receptacles may be spaced from one another by a distance selectedfrom 2.25 mm, 4.5 mm and 9 mm.

In another aspect, the system may include at least one controllerconfigured to modulate or pulsate the excitation sources and to select acomponent of a response signal from the detection elements based on themodulation or pulsation of the excitation sources. The at least onecontroller may be configured to perform Fourier transformation on theresponse signal to improve a signal-to-noise ratio of the system.

In another aspect, the system may include at least one controllerconfigured to modulate or pulsate the excitation sources at respectivefrequencies and to select a component of a response signal from thedetection elements based on the modulation or pulsation of theexcitation sources. For example, there may two excitation sources andone detector per receptacle, each of the excitation sources emitting atdifferent wavelengths. In such an embodiment, the at least onecontroller may be configured to operate the excitation sources atdifferent frequencies, and to demodulate a response signal based on thefrequencies. The at least one controller may be configured to determineinformation about different biochemical reactions occurring in the samereceptacle based on the demodulated response signal. The at least onecontroller may be configured to vary a current supplied to theexcitation sources to cause each of the excitation sources to emit atdifferent wavelengths at different times.

In another aspect, the present disclosure is directed to a sample holderfor use with a system that excites a sample received in the sampleholder with optical electromagnetic energy and detects opticalelectromagnetic energy returned by the sample, the sample holderincluding a wall member at least a portion of which provides an opticalpath between an exterior of the sample holder and an interior of thesample holder that filters a first band of wavelengths of opticalelectromagnetic energy in a range of optical electromagnetic energyemitted by an excitation source of a system and passes a second band ofwavelengths of optical electromagnetic energy detectable by a detectorof the system, where optical electromagnetic energy consists ofelectromagnetic energy between an ultraviolet and an infrared portion ofthe electromagnetic spectrum, inclusive.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the drawings, identical reference numbers identify similar elementsor acts. The sizes and relative positions of elements in the drawingsare not necessarily drawn to scale. For example, the shapes of variouselements and angles are not drawn to scale, and some of these elementsare arbitrarily enlarged and positioned to improve drawing legibility.Further, the particular shapes of the elements, as drawn, are notintended to convey any information regarding the actual shape of theparticular elements, and have been solely selected for ease ofrecognition in the drawings.

FIG. 1 is a diagrammatic view of an isothermal reaction and analysissystem including a receiver to receive sample holders, a thermal controlsubsystem, an excitation subsystem, a detection subsystem and ananalysis subsystem, according to one illustrated embodiment.

FIG. 2A is a top, front, left side isometric view of a receiver of anisothermal reaction and analysis system, according to one illustratedembodiment.

FIG. 2B is a top plan view of the receiver of FIG. 2A.

FIG. 2C is a front elevational view of the receiver of FIG. 2A.

FIG. 2D is a bottom plan view of the receiver of FIG. 2A.

FIG. 2E is a side elevational view of the receiver of FIG. 2A.

FIG. 3 is a diagrammatic view of an isothermal reaction and analysissystem including a receiver to receive sample holders, a thermal controlsubsystem, an excitation subsystem, a detection subsystem and ananalysis subsystem, according to another illustrated embodiment,particularly suited for use with non-elongated sample holders, forexample plates or other substrates.

DETAILED DESCRIPTION

In the following description, certain specific details are included toprovide a thorough understanding of various disclosed embodiments. Oneskilled in the relevant art, however, will recognize that embodimentsmay be practiced without one or more of these specific details, or withother methods, components, materials, etc. In other instances,well-known structures associated with temperature control including butnot limited to voltage and/or current regulators, excitation, detection,and/or signal and/or data processing, have not been shown or describedin detail to avoid unnecessarily obscuring descriptions of theembodiments.

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to.”

Reference throughout this specification to “one embodiment,” or “anembodiment,” or “in another embodiment” means that a particular referentfeature, structure, or characteristic described in connection with theembodiment is included in at least one embodiment. Thus, the appearanceof the phrases “in one embodiment,” or “in an embodiment,” or “inanother embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. It should also be notedthat the term “or” is generally employed in its sense including “and/or”unless the content clearly dictates otherwise.

The headings provided herein are for convenience only and do notinterpret the scope or meaning of the embodiments.

FIG. 1 shows a system 10 to support isothermal biochemical reactions,for example isothermal amplification nucleic acids and/or isothermalcell growth using inserted fluorescent proteins (FPs), for example BlueFPs or Green FPs, according to one illustrated embodiment.

The isothermal reaction and analysis system 10 includes a receiver 12having a plurality of receptacles 14a, 14b (only two called out in theFigure, collectively referenced as 14) formed therein. Each of thereceptacles 14 is sized to at least partially receive and support arespective one of a plurality of sample holders 16 a, 16 b, (only twoillustrated, collectively referenced as 16). In FIG. 1, one sampleholder 16 a is illustrated received in a respective receptacle 14 a andthe second sample holder 16 b is illustrated removed from the respectivereceptacle 14.

The isothermal reaction system 10 also includes a thermal controlsubsystem 20 operable to at least approximately maintain a temperatureat least proximate the sample holders 14. In particular, the thermalcontrol subsystem 20 maintains a temperature of the receiver 12approximately constant at a desired or set temperature, for a period oftime sufficient to perform an isothermal reaction on the samples 17 heldby the sample holders 16.

The thermal control subsystem 20 may include one or more heater elements22 operable to provide heat (indicated by arrow 24 a) to the receiver 12and/or one or more cooling elements operable to withdraw heat from thereceiver 12, in response to a difference between the detectedtemperature and at least one set temperature. In some embodiments theheater element 22 may recycle heat (indicated by arrow 24 b) from thereceiver 12. In some embodiments, the at least one heater element 22 isconductively thermally coupled to the receiver 12 to provide heat to thesample holders 14 via the receiver 12. In some embodiments, the one ormore cooling element is conductively thermally coupled to the receiver12 to withdraw heat from the sample holders 14 via the receiver 12. Asdiscussed below, in some embodiments the receiver 12 has a large thermalmass (i.e., specific heat capacity multiplied by mass) with respect tothe samples 17 to facilitate stable, constant and uniform temperatureoperation, which is useful in performing isothermal reactions. Theheater element 22 may take any of a variety of forms including resistorsor resistive elements, radiant elements or other sources of heat. Forexample, the heater element 22 may take the form of a thin-film KAPTONheater with an aluminum foil backing, commercially available from Minco.Such a heater element 22 may be directly positioned on or attached tothe receiver 12. The cooling elements may likewise take a variety offorms including refrigerator coils and/or heat sinks.

The thermal control subsystem 20 may further include an interface 26 toreceive an indication (indicated by arrow T_(REF)) of a desired or settemperature for the reactions. In some embodiments, the thermal controlsubsystem 20 may include a temperature or heater controller 28 toactively control the heater element 22 and/or a cooling element. Forexample, the thermal control subsystem 20 may include one or moretemperature sensors 30 operable to detect a temperature at leastproximate the sample holders 16 that is indicative of a temperature of abiochemical reaction within the sample holders 16. For example, thetemperature sensor 30 may detect a temperature of the receiver 12 at oneor more locations. The temperature or heater controller 28 may comparethe temperature detected by the temperature sensor 30 to the desired orset temperature TREF, and produce appropriate control signals (indicatedby arrow 32) to adjust operation of the heater element 22. Thetemperature or heater controller 28 may take a variety of formsincluding, but not limited to microcontrollers, microprocessors, digitalsignal processors (DSPs), application specific integrated circuits(ASICs), field programmable gate arrays (FPGAs), comparators, discretecircuit components, or the like. For example, the heater controller 28may take the form of an ON-OFF controller, for instance an ON-OFFcontroller commercially available from Minco as part no. Heaterstat,CT198-1007R25L1. In some embodiments, heat 24 is provided through thereceiver 12 via one or more channels 34 formed in the receiver 12. Thismay advantageously spread the heat more evenly throughout the receiver12.

The isothermal reaction system 10 may include an excitation subsystem 40operable to excite each of a number of samples 17 contained inrespective ones of a plurality of the sample holders 16 withelectromagnetic energy while the sample holders 16 are received andsupported in the receptacles 14 of the receiver 12. The isothermalreaction system 10 may also include a detection subsystem 42 operable todetect emission of electromagnetic energy from the samples 17 containedin a respective ones of the sample holders 16 while the sample holders16 are received and supported in the receptacles 14 of the receiver 12.

The excitation subsystem 40 includes a first plurality of excitationsources 44 a (collectively 44, only one called out for sake of clarityof illustration). Each of the first plurality of excitation sources 44is positioned to direct the electromagnetic energy toward at least aportion of a respective one of the sample holders 16 received in arespective one of the receptacles 14 of the receiver 12.

The excitation subsystem 40 may optionally include a second plurality ofexcitation sources 46 a (collectively 46, only one illustrated, removedfor sake of clarity of illustration). Each of the second plurality ofexcitation sources 46 is positioned to direct the electromagnetic energytoward at least a portion of a respective one of the sample holders 16received in a respective one of the receptacles 14 of the receiver 12.The electromagnetic energy directed by the second plurality ofexcitation sources 46 may be of a different wavelength or differentbands of wavelength than the electromagnetic energy directed by thefirst plurality of excitation sources 44. In some embodiments, at leastone filter 48 (only one illustrated for sake of clarity of drawing) ispositioned between at least one excitation source 44, 46 and the atleast one of the sample holders 16 when the sample holder 16 is receivedby a respective receptacle 14 of the receiver 12. For example, thefilter 48 may take the form of an amber cast acrylic sheet (e.g.,PLEXIGLASS), which forms a very sharp long pass optical filter atapproximate 550 nm.

The excitation sources 44, 46 may, for example, take the form of lightemitting diodes (LEDs). The LEDs may be operable to emit electromagneticenergy in at least an optical portion of the electromagnetic spectrumbetween an ultraviolet portion of the electromagnetic spectrum and aninfrared portion of the electromagnetic spectrum, inclusive. Forexample, the first plurality of excitation sources 44 may include anumber of LEDs which are operable to emit electromagnetic energy in afirst band, and the second plurality of excitation sources 46 mayinclude a number of LEDs which are operable to emit electromagneticenergy in a second band, different than the first band. The bands may bedistinct, and these distinct bands may, or may not overlap. Increasinglypowerful LEDs in multiple colors with small formats are becomingcommercially common. For example, a suitable LED may be commerciallyavailable form Sunbrite as SSP-01TWB7UWB12 12V 470 nm Blue LED.Alternatively, all excitations sources 44, 46 may emit at the samewavelengths, and filters used to produce different excitation from therespective excitation sources 44, 46.

In particular, the excitation sources 44, 46 may be positioned at leastpartially in respective ones of a number of passages 50 (only one calledout in FIG. 1, for sake of clarity of illustration) formed in thereceiver 12. A portion of each of the excitation sources 44, 46 isproximate (e.g., less than 2 mm, or less than about 1 mm) respectiveones of the receptacles 14. This locates the excitation sources 44, 46close to the sample holders 16, for example within 2 mm or 1 mm,improving analysis and, for at least some methods of amplificationmaking analysis possible where it might not otherwise be possible withconventional excitation sources. In at least one embodiment, each of theexcitation sources 44, 46 are positioned with respect to the receiver 12to direct the electromagnetic energy toward only a base portion 52 of asidewall of the respective sample holder 16 when the sample holder 16 ispositioned in the respective one of the receptacles 14. This may beadvantageous since the sample 17 is likely to collect at the bottom ofthe sample holder 16, under the influence of gravity.

The excitation subsystem 40 may also include an excitation controller54, operable to control the operation of the excitation sources 44, 46via appropriate signals (indicated by arrow 56). For example, theexcitation controller 54 may control when the excitation sources 44, 46emit electromagnetic energy. Additionally, or alternatively, theexcitation controller 54 may control the duration during which theexcitation sources 44, 46 emit electromagnetic energy. Additionally, oralternatively, the excitation controller 54 may control the level ormagnitude of electromagnetic energy emitted by the excitation sources44, 46. Additionally, or alternatively, the excitation controller 54 maycontrol the wavelength or band of wavelengths emitted by the excitationsources 44, 46. The signals 56 may take any of a variety of formsincluding, but not limited to currents, voltages, impedances, light,radio frequency, and/or microwave signals.

The detection subsystem 42 may include one or more detectors 60 a(collectively 60, only one called out for sake of clarity ofillustration). Each of the detectors 60 is positioned to detectelectromagnetic energy emitted by the sample in a sample holder 16 whilethe sample holder 16 is received in a respective one of the receptacles14 of the receiver 12. In some embodiments, at least one filter 62 ispositioned between the detectors 60 and the respective sample holder 16when the sample holder 16 is received by the respective receptacle 14 ofthe receiver 12. The filter 62 may, for example, substantially blockwavelengths emitted by the excitation sources 44, 46, whilesubstantially passing wavelengths emitted by the samples 17 (e.g.,fluorescence) in response to the excitation. The filter 62, may forexample, take the form of a 532 nm optical band pass filter.

The detectors 60 of the detection subsystem 42 may, for example, takethe form of photodiodes, photodetectors, or other electromagneticsensitive devices. The detectors 60 may, for example, be responsive toelectromagnetic energy in at least of a portion of the electromagneticspectrum between an ultraviolet portion of the electromagnetic spectrumand an infrared portion of the electromagnetic spectrum, inclusive. Thedetectors 60 may, for example, include a number of photodiodes that areresponsive to electromagnetic energy in a portion of the electromagneticspectrum that is different from the portion of the electromagneticspectrum in which the excitation sources 44, 46, for example LEDs. Avariety of photodiodes may be suitable. For example, a single chipdesign photodiode with a built in amplifier may be commerciallyavailable from Texas Instruments as OPT101. A photodiode with threecolor detection (RGB) and individual outputs for each color may becommercially available from Hamamatsu as S9702. A line-of-sight orfield-of-view of the photodiodes 60 may be arranged perpendicular to aprincipal direction or axis of emission of the excitation sources 44,46. Such may prevent the emission by the excitation sources 44, 46 frominterfering with or creating excessive noise for the detectorsphotodiodes 60, reducing or eliminating the need for filtering.

In particular, the detectors 60 may be positioned at least partially inrespective ones of a number of openings 64 (only one called out in FIG.1, for sake of clarity of illustration) formed in a bottom or lower face63 of the receiver 12. A portion of each of the detectors 60 isproximate (e.g., less than 2 mm, or less than about 1 mm) respectiveones of the receptacles 14. This locates the detectors 60 close to thesample holders 16, for example within 2 mm or 1 mm, improving detectionand, for at least some methods of amplification, making analysispossible where it might not otherwise be possible with conventionalexcitation sources and detectors with or without lenses, reflectors orother optical elements. In at least one embodiment, each of thedetectors 60 are positioned with respect to the receiver 12 to beproximate the base portion 52 of the respective sample holder 16 whenthe sample holder 16 is positioned in the respective one of thereceptacles 14. This may be advantageous since the sample 17 is likelyto collect at the bottom of the sample holder 16, under the influence ofgravity. Where the sample holder 16 is not elongated, for example a flatplate or tape, the detectors 60 may alternatively be positioned abovethe receptacles 14 of the receiver 12.

The detector subsystem 42 may include a detection controller 66,operable to receive and process data detected by the detectors 60. Thedetection controller 66 may receive signals encoding data or otherinformation (indicated by arrow 67) from the detectors 60. For example,the detection controller 66 may transform analog signals to digitalsignals. Also for example, the detection controller 66 may otherwisepreprocess signals, for instance performing electronic filtering. Thesignals 67 may take any of a variety of forms including, but not limitedto currents, voltages, impedances, light, radio frequency, and/ormicrowave signals. The detection controller 66 may take a variety offorms, for example hardware, software stored in a storage device,firmware or a combination of such. The detection controller 66 may, forexample, be implemented by a general purpose computer executinginstructions that cause the general purpose computer to function as aspecial purpose machine. The detection controller 66 may be implementedas a microcontroller, microprocessor, DSP, ASIC and/or FPGA, or asdiscrete electrical and/or electronic components.

The detector subsystem 42 may include an analysis subsystem 68. Theanalysis subsystem 68 may receive raw or preprocessed data orinformation (indicated by arrow 69) from the detection controller 69.The analysis subsystem 68 may analyze the data 69 according to a varietyof known protocols and protocols to be developed. For example, theanalysis subsystem 68 may analyze the data 69 for matches betweennucleic acids. The analysis subsystem 68 may take a variety of forms,for example hardware, software stored in a computer-readable storagedevice 65, firmware or a combination of such. The analysis subsystem 68may, for example, be implemented by a general purpose computer executinginstructions that cause the general purpose computer to function as aspecial purpose machine. The analysis subsystem 68 may be implemented asa microcontroller, microprocessor, DSP, ASIC and/or FPGA, or as discreteelectrical and/or electronic components. In some embodiments, theanalysis subsystem 68 or its functions may be part of, or implemented bythe detection controller 66. In other embodiments, the detectioncontroller 66 or its functions may be part of, or implemented by theanalysis subsystem 68. Preprocessing may, for example include real-timesignal averaging, followed by non-causal zero phase digital postprocessing digital filtering. In some embodiments, a differentialinstrumentation amplifier may be used to interface with a universalserial bus (USB) analog digital converter.

FIGS. 2A-2E show a receiver 12, according to one illustrated embodiment.The receiver 12 may take the form of an elongated metal bar. Thereceiver 12 may, for example, be formed from aluminum.

In the embodiment illustrated in FIGS. 1 and 2A-2E, the receiver 12 hasthe plurality of receptacles 14 formed in an upper face 70 of the metalbar. Each of the receptacles 14 has a diameter that is sized to closelyreceive a diameter of a respective one of the sample holders 16. Thereceiver 12 may include a pair of angled faces 72 a, 72 b, each formingan obtuse angle 74 a, 74 b between with the upper face 70. Each of theangled faces 72 a, 72 b may have a respective set of passages 50 a, 50 b(only four of each called out in FIGS. 2B-2D). Each of the passages 50a, 50 b extends from an exterior of the receiver 12 to an interior ofthe receiver 12, providing communication with a respective one of thereceptacles 14. Each of the passages 50 a, 50 b may receive at least aportion of a respective one of the excitation sources 44 a, 44 b. Asnoted above, the receiver 12 has a large thermal mass. For example, thereceiver 12 may have a thermal mass ten times greater than the thermalmass of the samples 17.

FIG. 3 shows an isothermal reaction and analysis system 100, accordingto another illustrated embodiment, particularly suited for use withnon-elongated sample holders, for example plates or other substrates.Structures which are identical or similar to previously describedembodiments are called out with the same reference numbers arepreviously used.

In particular, a receiver 112 has the plurality of receptacles 114 (onlyone called out in FIG. 3, for sake of clarity of illustration) formed inan upper face 170 of the metal bar. Each of the receptacles 114 has adiameter that is sized to closely receive a diameter of a respective oneof the sample holders. The sample holders may, for example, take theform of wells 116 (only one called out in FIG. 3 for sake of clarity ofillustration) formed on a plate, microfluidic cartridge or othersubstrate 117. Plates or substrates 117 may, for example, be compatiblewith ANSI/SBS standards 1-2004, 2-2004, 3-2004 and 4-2004. For example,the receptacles 14, 114 may are spaced from one another by a distanceselected from 2.25 mm, 4.5 mm and 9 mm.

The receiver 12 may further have a number of passages 150 (only onecalled out in FIG. 3 for sake of clarity of illustration) formed in alower face 163 that is opposed to the upper face 170. Each of thepassages 150 extends from an exterior of the metal bar to an interior incommunication with a respective one of the receptacles 114. Excitationsources, for example LEDs 144 (only one called out in FIG. 3 for sake ofclarity of illustration) are positioned at least partially in respectiveones of the passages 150 with a portion of each of the LEDs 144proximate respective ones of the receptacles 114. This locates the LEDs144 proximate the wells or sample holders 116 when the wells or sampleholders 116 are received in the receptacles 114. Such may facilitateanalysis, or make analysis possible for some amplification processeswhere such would not otherwise have been possible using existingexcitation sources.

Detectors, for example, photodiodes 160 may be positioned relativelyabove the receiver 112, aligned with respective receptacles 114. Thephotodiodes 160 may, for example, be carried by a common substrate, forexample a circuit board 171. A line-of-sight or field-of-view of thephotodiodes 160 may be arranged collinearly or coaxially or parallel toa principal direction or axis of emission 173 (only one shown forclarity of illustration) of the LEDs 144. Filters 62 may be positionedbetween the photodiodes 160 and the wells 116 when the wells 116 arereceived in the receptacles 114. Such may prevent the emission by theLEDs 144 from interfering with or creating excessive noise for thephotodiodes 160.

Some embodiments, the system 10, 100 may take the form of a binaryformat device. In such embodiments, some of the receptacles 14, 114 mayreceive sample holders 16, 116 holding a reference sample while otherreceptacles 14, 114 may receive sample holders 16, 116 holding a targetsample undergoing reaction and/or analysis. In such an embodiment thesystem 10, 100 may provide a binary response (e.g., YES/NO,POSITIVE/NEGATIVE). The reference sample may, for example, have acomplete amplification mixture without a trigger. A reference signalfrom the reference sample may be automatically subtracted from thesignal representing the unknown sample, for example via an instrumentamplifier integrated circuit. The instrument amplifier integratedcircuit may turn ON LEDs or other indicators if a voltage difference(i.e., amplification) is detected. 8-pin photodiodes may be coupled viarespective 10-pin vertical DIP sockets, allowing the heater or a coolingelement to be mounted directly to a main circuit board in the binaryformat device.

In some embodiments, the excitation sources 44, 46, 144 may be modulatedin order to enhance the signal-to-noise ratio at the detectors 16, 116,reduce the effect of background noise, and/or enable multiplexing. Forinstance, excitation controller 54 may pulsed (ON/OFF) or modulated LEDs144 at a well defined frequency, and the detected signal obtained by thephotodiode 160 or other detector may be Fourier transformed, for exampleby detection controller 66 or analysis subsystem 68, to identify thecomponent of the response signal at the same frequency as that of theexcitation source 44, 46, 144. Such can be used, for example, toeliminate noise from ambient illumination. Similarly, selectingwell-defined modulation or pulsing frequencies for the excitationsources 44, 46, 144, and extracting the component of the response at thesame frequency enables the signal-to-noise ratio to be improved, evenwhere noise is characterized as having a broad spectrum of frequencies.

In some embodiments, multiplexing may be achieved with respect to theexcitation sources 44, 46 and/or detectors 60 using different pulsing ormodulation frequencies. Such may allow various frequency components ofthe detector signal to characterize the response of the sample todifferent excitation source/filter combinations. This may allow two ormore labels (e.g., fluorescent tags or markers) to be detected using asingle detector 60, reducing the cost of the device. Each definedfrequency component corresponds to the excitation of the appropriatefluorophore by its specific excitation source/filter combination. Forexample, the excitation controller 54 may operate a first excitationsource 44 associated with a receptacle 14 at a first frequency to emitat a first wavelength, and operate a second excitation source 46associated with the same receptacle 14 at a second frequency to emit ata second wavelength, different from the first wavelength. The sample 71in the receptacle 14 may have two or more biochemical reactions takingplace, a first reaction which produces fluorescence in response to thefirst wavelength and the second reaction which produces fluorescence inresponse to the second wavelength. A single detector 60 produces aresponse signal in response to the fluorescence that results from boththe first and second wavelengths of excitation. The detection controller66 or analysis subsystem 68 may demodulate the response signal based onthe first and the second frequencies, to determine information about thefirst and the second biochemical reactions (e.g., whether reactionsoccurred, to what extent reactions occurred, products of reactions,etc.). In some embodiments, the excitation controller 54 may operate allof the first plurality of excitations sources 44 at the first frequencyand all of the second plurality of excitation sources 46 at the secondfrequency. In other embodiments, the excitation controller 54 mayoperate each of excitations sources 44, 46 at a respective frequency. Insome embodiments, the excitation controller 54 may vary a currentsupplied to the excitation sources to vary the emission wavelength ofthe excitation source. Such may advantageously reduce the total numberof excitations sources without reducing the amount of information aboutthe biochemical reactions that may be derived.

In some embodiments, the system 10, 100 may be used to measure thegrowth of cells that express various fluorescent proteins.

In one embodiment, the system 10, 100 may be constructed by assembly ofelectronic components and computer numeric controlled (CNC) machining. A96 well aluminum plate (Biosmith, 81001), designed to accommodate 200 μltubes, may be cut down in size to provide a row of 8 wells. Two DELRINplastic parts may be machined (e.g., CNC machined) to provide separateholders for the LED and the combination of the photodetector and opticalfilter. Each DELRIN part has clearance holes for 4-40 screws, which mateto the aluminum 8 well vessel. Two holes may be tapped in the aluminum 8well vessel to accommodate 4-40 screws, which align with the DELRINclearance holes. A 10 Mf2 feedback resistor is connected to the outputand input pins of the OPT101 photodetector from Texas Instruments. Thephotodetector may be powered with two 9 Volt batteries for positive andnegative supply voltages. Voltage regulators, LM7808 and LM7908 regulatethe voltage to the photodetector at positive and negative 8 Volts,respectively. The output of the OPT101 photodetector may be connected tothe differential inputs of an INA 118P instrumentation amplifier fromTexas Instruments. The gain of the amplifier may be set to 51 using a 1kΩ resistor. The output from the OPT101 is connected to the single endedinputs of channel 0 of a USB analog to digital converter (PMD1208LS,Measurement and Computing). A separate 9 Volt battery may supply voltageto an LM7808 voltage regulator, which supplies current to an LED inseries with a 470Ω resistor. Holes in the aluminum 8 well vessel may bemachined to allow the fluorescence excitation light to penetrate atransparent 200 microliter tube and to allow the emission light to bedetected by a photodetector. A thin-film Kapton heater with an aluminumfoil backing, purchased from Minco, may be adhered to the side wall ofthe aluminum 8 well vessel. The heater may be electrically connected toa resistance-matched, Minco on-off controller (Heaterstat,CT198-1007R25L1), which is connected to an AC to DC transformer. Thecontroller may be adjusted to provide a temperature set point requiredby the reaction. The generic 200 μl tube (Biorad) may be filled with theEXPAR amplification reaction and Sybr Green II. An oligonucleotidesequence to initiate the EXPAR reaction may be added to the sampleholder (reaction tube), at 0 degrees C., external to the device. Thesample holder (reaction tube) may be placed in the appropriatereceptacle of the aluminum 8 well/receptacle vessel. A LABVIEW programmay be initiated to average the incoming, sub-volt signal for onesecond. The result may be plotted and stored to a text file. Theresulting text file may be processed by Matlab with a non causal zerophase distortion digital filter routine using a twenty second signalprocessing window.

In some embodiments, the sample holders 16, 116 may, for example, takethe form of Society for Biomolecular Screening (SBS) standard formattubes. Such tubes are typically low cost, and disposable. In someembodiments, the sample holders 16, 116 may take the form of wellsformed in plates or other substrates, for example plates or substratesthat are compatible with ANSI/SBS standards 1-2004, 2-2004, 3-2004 and4-2004. In some embodiments, the sample holders 16, 116 may take otherforms, with specific geometries and material properties to facilitatereaction, assaying or other functions.

At least a portion of each of the sample holders 16, 116 comprises amaterial that passes at least a portion of electromagnetic energyemitted by the excitation sources 44, 46, 144 of the excitationsubsystem 40. At least a portion of each of the sample holders 16, 116comprises a material that passes at least a portion of theelectromagnetic energy emitted by samples held in the sample holders 16,116 in response excitation by electromagnetic energy from the excitationsources 44, 46, 144. In this respect, it is noted that theelectromagnetic energy emitted by the samples (e.g., florescence) may bedifferent or have a different set or band of wavelengths than theelectromagnetic energy emitted by the excitation sources 44, 46, 144 ofthe excitation subsystem 40. In particular, at least a bottom portion ofthe sample holder 16, 116 provides an optical path between an exteriorof the sample holder 16, 116 and an interior 200 of the sample holder16, 116. Typically, all or substantially all of the sample holders 16,116 will be formed of or including a coating of a same material, withouta transition of materials along the length or around a perimeter of thesample holders 16, 116. The sample holder may be formed from thefiltering material or with the filtering material included, or thefiltering material may take the form of one or more coatings or layersapplied on an exterior or interior, to become an integral part of thesample holder 16, 116.

In some embodiments, at least a portion of each of the sample holders16, 116 comprises a material that additionally filters out a portion ofelectromagnetic energy emitted by the excitation sources 44, 46, 144 ofthe excitation subsystem 40. Such may advantageously eliminate the useof separate filters 48, 62. For example, such material may substantiallyblock wavelengths that would interfere with the detectors 60, whilepassing wavelengths that excite the samples. Such may additionally, oralternatively protect the contents of the sample holders 16, 116 fromambient electromagnetic energy.

In some embodiments, at least a portion of the sample holders 16, 116comprises a material that additionally, or alternatively, filters out aportion of the electromagnetic energy emitted by the samples held in thesample holders 16, 116 in response excitation by electromagnetic energyfrom the excitation sources 44, 46, 144 of the excitation subsystem 40.In this respect, it is noted that the material may filter out a firstwavelength, set or band of wavelengths emitted by the excitationsubsystem and may filter out a second wavelength, set or band ofwavelengths emitted by samples, where the first and the secondwavelengths, set or band of wavelengths are different from one another,which first and second wavelengths, sets or bands of wavelengths may, ormay not, be overlapping.

The sample holders 16 may advantageously be sealed during use,preventing contamination. In particular, the sample holder 16 may havean upper portion that is selectively sealable, for example via a cap202, or adhesive, epoxy or via the sealing of edges forming a perimeterof an opening via heat and/or application of force.

The disclosed embodiments provide a low cost, high speed and effectivesolution to performing biochemical reactions such as isothermalamplification and/or analysis of the results of such reactions. Thedisclosed embodiments further provide a reaction and/or analysis devicethat is light weight, has a small form factor, for example beinghandheld, and hence is portable. The disclosed embodiments are capableof operating with existing sample holders, avoiding the need to stockmultiple types of sample holders or the need to replace existing stocksof sample holders. The disclosed embodiments advantageously permitexcitation and detection to occur through the sidewalls of a sampleholder, eliminating the need for flat bottom wells or flat lids.Further, the disclosed embodiments allow real time collection and/oranalysis of data during biochemical reactions. Such may permitquantification, rather than simply detection of a presence or absence ofa reaction product. The disclosed designs allow the elimination of lightcollecting optics due to the small footprint and positioning of theexcitation sources and/or detectors. The disclosed designsadvantageously allow simultaneous detection of fluorescence in multiplewells, increasing throughput of the system. The disclosed designs alsopermit the use of sample holders with relatively small surface-to-volumeratios, compared with PCR thermal cycling devices. The disclosed designsalso allow easy integration since excitation and detection may beperformed through the base portion of the sidewall of the sampleholders.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety.

The above description of illustrated embodiments, including what isdescribed in the Abstract, is not intended to be exhaustive or to limitthe embodiments to the precise forms disclosed. Although specificembodiments of and examples are described herein for illustrativepurposes, various equivalent modifications can be made without departingfrom the spirit and scope of the disclosure, as will be recognized bythose skilled in the relevant art. The teachings provided herein of thevarious embodiments can be applied to isothermal reactors and/oranalysis devices, not necessarily the exemplary nucleic acidamplification and analysis device generally described above.

For instance, the foregoing detailed description has set forth variousembodiments of the devices and/or processes via the use of blockdiagrams, schematics, and examples. Insofar as such block diagrams,schematics, and examples contain one or more functions and/oroperations, it will be understood by those skilled in the art that eachfunction and/or operation within such block diagrams, flowcharts, orexamples can be implemented, individually and/or collectively, by a widerange of hardware, software, firmware, or virtually any combinationthereof. In one embodiment, the present subject matter may beimplemented via Application Specific Integrated Circuits (ASICs).However, those skilled in the art will recognize that the embodimentsdisclosed herein, in whole or in part, can be equivalently implementedin standard integrated circuits, as one or more computer programsrunning on one or more computers (e.g., as one or more programs runningon one or more computer systems), as one or more programs running on oneor more controllers (e.g., microcontrollers) as one or more programsrunning on one or more processors (e.g., microprocessors), as firmware,or as virtually any combination thereof, and that designing thecircuitry and/or writing the code for the software and or firmware wouldbe well within the skill of one of ordinary skill in the art in light ofthis disclosure.

In addition, those skilled in the art will appreciate that themechanisms of taught herein are capable of being distributed as aprogram product in a variety of forms, and that an illustrativeembodiment applies equally regardless of the particular type of signalbearing media used to actually carry out the distribution. Examples ofsignal bearing media include, but are not limited to, the following:recordable type media such as floppy disks, hard disk drives, CD ROMs,digital tape, and computer memory; and transmission type media such asdigital and analog communication links using TDM or IP basedcommunication links (e.g., packet links).

The various embodiments described above can be combined to providefurther embodiments. All of the commonly assigned US patent applicationpublications, US patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, including butnot limited to U.S. patent application Ser. No. 14/307,253, filed Jun.17, 2014, U.S. patent application Ser. No. 13/415,709, filed Mar. 8,2012; U.S. patent application Ser. No. 12/052,950, filed Mar. 21, 2008;and U.S. provisional patent application Ser. No. 60/896,452, filed Mar.22, 2007 are incorporated herein by reference, in their entirety.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1-24. (canceled)
 25. A system to support isothermal nucleic acidamplification reactions in a plurality of samples, the systemcomprising: a receiver having a plurality of receptacles formed therein,each of the receptacles sized to at least partially receive and supporta respective one of a plurality of sample holders wherein the receiverhas a large thermal mass relative to a respective thermal mass of thesamples; an excitation subsystem including at least a first plurality ofexcitation sources, each of the excitation sources having a respectiveaxis of emission and positioned to direct electromagnetic energy towardat least a portion of a respective one of the sample holders received ina respective one of the receptacles of the receiver, the excitationsubsystem operable to emit electromagnetic energy with a firstwavelength sufficient to excite fluorescence in each of the plurality ofsamples contained in respective ones of the plurality of the sampleholders while the sample holders are received and supported by thereceiver; a detection subsystem that includes a plurality of detectors,each of the detectors having a respective line of sight and positionedto detect fluorescence emission of electromagnetic energy at awavelength that is different from the first wavelength and which isemitted by a respective sample contained in a respective one of thesample holders that are received and supported by the receiver, each ofthe detectors positioned such that the respective line of sight of thedetector and the respective axis of emission of the respectiveexcitation source are arranged at an angle to reduce the detection ofemission directly from the excitation sources; and a thermal controlsubsystem operable to at least approximately maintain an at leastapproximately uniform temperature of the receiver at least approximatelyand said temperature constant for a period of time sufficient to performan isothermal nucleic acid amplification on the samples.
 26. The systemof claim 25 wherein the excitation subsystem includes a second pluralityof excitation sources, each of the excitation sources of the secondplurality of excitation sources positioned to direct the electromagneticenergy toward at least a portion of a respective one of the sampleholders received in a respective one of the receptacles of the receiver,the electromagnetic energy directed by the excitation sources of thesecond plurality of excitation sources being of a wavelength that isdifferent than the wavelength of the electromagnetic energy directed bythe excitation sources of the first plurality of excitation sources. 27.The system of claim 26 wherein each of the excitation sources ispositioned completely within a respective passage in the receiver todirect the electromagnetic energy toward only a base portion of therespective sample holder when the sample holder is positioned in therespective one of the receptacles, the base portion at least proximate abottom of the sample holder when supported by the receiver.
 28. Thesystem of claim 25, further comprising: at least one filter positionedbetween at least one excitation source and the at least one of thesample holders when the at least one of the sample holders is receivedby the respective receptacle of the receiver.
 29. The system of claim25, further comprising: at least one filter positioned between at leastone of the detectors and the at least one of the sample holders when theat least one of the sample holders is received by the respectivereceptacle of the receiver, the at least one filter operative to filterat least a portion of the electromagnetic energy emitted by at least oneof the excitation sources.
 30. The system of claim 25 wherein thethermal control subsystem includes at least one temperature sensoroperable to detect a temperature at least proximate the sample holdersthat is indicative of a temperature of a biochemical reaction within thesample holders and at least one heater element operable to provide heatto the receiver in response to a difference between the detectedtemperature and at least one set temperature.
 31. The system of claim 30wherein the at least one heater element is conductively thermallycoupled to the receiver to provide heat to the sample holders via thereceiver.
 32. The system of claim 25 wherein the detectors of thedetection subsystem include a number of photodiodes that are responsiveto electromagnetic energy in a portion of the electromagnetic spectrumbetween an ultraviolet portion of the electromagnetic spectrum and aninfrared portion of the electromagnetic spectrum, inclusive.
 33. Thesystem of claim 25 wherein the excitation subsystem is operable to emitelectromagnetic energy in a portion of the electromagnetic spectrumbetween an ultraviolet portion of the electromagnetic spectrum and aninfrared portion of the electromagnetic spectrum, inclusive.
 34. Thesystem of claim 25 wherein each of the sample holders comprises amaterial that filters out a portion of electromagnetic energy emitted bythe excitation subsystem and passes a portion of electromagnetic energyemitted by the excitation subsystem.
 35. The system of claim 25 whereinthe receptacles are spaced from one another by a distance selected from2.25 mm, 4.5 mm and 9 mm.
 36. The system of claim 25, furthercomprising: at least one controller communicatively coupled to providecontrol signals to at least one of modulate or pulsate the excitationsources and to select a component of a response signal from thedetectors based on the modulation or pulsation of the excitationsources.
 37. The system of claim 36 wherein in use the at least onecontroller performs a Fourier transformation on the response signal toimprove a signal-to-noise ratio of the system.
 38. The system of claim25, further comprising: at least one controller communicatively coupledto provide respective control signals to at least one of modulate orpulsate the excitation sources at respective frequencies and to select acomponent of a response signal from the detectors based on themodulation or pulsation of the excitation sources.
 39. The system ofclaim 25 wherein there are at least two excitation sources and onedetector per receptacle, where each of at least two of the excitationsources per receptacle emit at different wavelengths, and furthercomprising: at least one controller communicatively coupled to operate afirst one of the two excitations sources of a first receptacle at afirst frequency and a second one of the excitation sources of the firstreceptacle at a second frequency, different from the first frequency,and to demodulate a response signal from the detector based on the firstand the second frequencies.
 40. The system of claim 39 wherein in usethe at least one controller determines at least one piece of informationabout a first isothermal nucleic acid amplification reaction occurringin the first receptacle and at least one piece of information about asecond isothermal nucleic acid amplification reaction occurring in thefirst receptacle based on the demodulated response signal.
 41. Thesystem of claim 25, further comprising: at least one controllercommunicatively coupled to vary a current supplied to the excitationsources to cause each of the excitation sources to emit a firstwavelength at a first time and a second wavelength different from thefirst wavelength at a second time.
 42. The system of claim 25 whereinthe isothermal nucleic acid amplification reaction is an isothermalnucleic acid amplification reaction chosen from the group consisting ofstrand displacement amplification, self-sustained replication, nucleicacid sequence based amplification, transcription mediated amplification,Q beta replicase amplification, loop mediated isothermal Amplification,and exponential amplification reaction.
 43. The system of claim 25wherein each of the first plurality of excitation sources is positionedto direct electromagnetic energy toward only one of the plurality ofreceptacles.